Targeting Regulation of CHPT1 Gene Expression by miR-33 in Chicken (Gallus gallus)
SHAO Fang1, 2, *, WANG Xing-Guo2, 3, YU Jian-Feng2, LI Hui4, GU Zhi-Liang2, *
1 The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou 213003, China; 2 School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China; 3 Jiangsu Institute of Poultry Science, Yangzhou 225125, China; 4 College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, China
Abstract：In addition to the regulation of lipid metabolism, miR-33 is also reported to be involved in glucose metabolism, inflammatory response, and cell cycle. However, the role of the chicken (Gallus gallus) miR-33 and its relationship with the predicted target gene choline phosphotransferase 1 (CHPT1) are still unclarified. The present study was conducted to investigate whether CHPT1 was the target gene of miR-33. The bioinformatics methods were used to predict the target genes of miR-33, and 378 genes from 3'-UTR database were predicted and CHPT1 gene was included. Then, the miR-33 overexpression vector and CHPT1 luciferase reporter vector were constructed and co-transfected into the mouse (Mus musculus) C2C12 myoblast cell, dual-luciferase reporter assay showed that the expression of luciferase reporter gene linked to the 3'-untranslated region of CHPT1 mRNA was down-regulated by miR-33 overexpression in C2C12 cells (P<0.01). Furthermore, the down-regulation was completely abolished when the predicted miR-33 target site in CHPT1 3'-UTR was mutated. Then mir-33 antagonists LNA (locked nucleic acid)-antimiR-33 was designed and synthetised, after transfecting chicken primary liver cells, the expression level of miR-33 decreased by 44%, while CHPT1 mRNA increased with a certain degree. By qRT-PCR, it was found that miR-33 highly expressed in muscular stomach and heart muscle, and the miR-33 expression in muscular stomach was significantly different from that in spleen, kidney, brain and glandular stomach (P<0.05), and very significantly different from that in liver and thigh muscle (P<0.01). In liver and abdomen fat tissue of 4 week lean and fat line chicken, miR-33 was significantly higher in the fat line than that in the lean line (P<0.05). The CHPT1 expression in the abdominal fat tissues was significantly higher in fat line chicken than that in lean line chicken (P<0.05). The above data indicate that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of CHPT1. The present study provides new clues for lipid synthesis.
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